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Requirement for RORγ in Thymocyte Survival and Lymphoid Organ Development
Zuoming Sun, Derya Unutmaz, Yong-Rui Zou, Mary Jean Sunshine, Alessandra Pierani, Susan Brenner-Morton, Reina E. Mebius, and Dan R. Littman

Supplementary Material

Supplemental Figure 1. Generation of RORγ-/-mice by targeted mutagenesis. (A) Genomic organization of the wild-type allele, the targeting construct, and the inactivated allele are illustrated in the top, middle, and bottom panels, respectively. A 200-bp fragment containing the exon encoding the DNA-binding domain of RORγ was replaced with the 1.2-kb neomycin resistance gene and flanking plasmid DNA (stippled filling). H, Hind III; B, Bam HI; K, Kpn I. (B) Southern blot analysis of genomic DNA from littermates derived from a heterozygous intercross. Following digestion of genomic DNA with Kpn I, the blots were probed with the 5´ DNA fragment indicated in (A). (C) Northern blot analysis of RORγ expression in different tissues from wild-type (+/+) and RORγ-null mice (-/-), and from thymus of RAG1-/-mice. A full-length RORγ cDNA was used as probe. (D) Western blot analysis of RORγ expression in wild-type and mutant thymus. Equal numbers of cells were loaded in each lane.

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Methods: A sequence corresponding to the DNA binding domain in the murine RORγ cDNA was used to screen a mouse 129Sv/J genomic library. Positive clones were mapped and partially sequenced, and one clone containing an exon that encoded the entire DNA binding domain was identified. The targeting vector for the inactivation of the RORγ gene was constructed using the PL2-Neo vector by replacing the exon encoding the DNA-binding domain of RORγ with the neomycin resistance gene. An HSV-TK selection marker gene was inserted at the end of the long arm, which consisted of a 9-kb Bam HI fragment. The 1.2-kb short arm fragment was amplified by PCR and cloned between Cla I and Xba I in the polylinker of the PL2-Neo vector. E14 ES cells were transfected with linerized targeting vector and G418-resistant and gancyclovir-resistant clones that had undergone homologous recombination were identified by PCR using oligonucleotides 5´-CGACTGCATCTGCGTGTTCGAATTC-3´ and 5´-GTTCTGGTTCCCCAAGTTCAGGATG-3´. Homologous recombination was also confirmed by Southern blot analysis after digestion of ES cell DNA with Kpn I and hybridization with a probe located upstream of the long arm. The targeting vector for the inactivation of the RORγ gene was constructed using the PL2-Neo vector by replacing the exon encoding the DNA-binding domain of RORγ with the neomycin resistance gene. Out of 150 drug-resistant clones, three clones were positive for homologous recombination. Two of the clones were used to generate chimeric founder mice by microinjection into C57BL/6J blastocysts.

Supplemental Figure 2. TCR and FasL do not mediate apoptosis in RORγ-deficient mice. TCR-Cα-/-or gld/gld mice were crossed to RORγ-/- mice to obtain double null animals. Survival of freshly isolated thymocytes, cultured in medium for various time intervals, is shown. The level of apoptosis was determined by FACS analysis of cells stained with Annexin V and propidium iodide. Percent of cells that remained Annexin V- PI- is indicated for each time point.

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Supplemental Figure 3. Analysis of RORγ expression in CD4+CD3-CD45+ cells. Top panel: CD4+CD3-CD45+ cells from day 0 the mesenteric lymph nodes were sorted by FACS. A series of dilutions of the cells (from 750 to 1.2) were used as template in RT-PCR analysis of RORγ expression. DP thymocytes from adult thymus and CD62hiCD44lo cells from adult lymph nodes were also sorted by FACS and used as positive and negative controls for RORγ expression. An equivalent number of cells was used in each assay. Middle panel: 1 μl of RT-PCR product from the top panel was used as template for 15 more cycles of PCR amplification with RORγ-specific primers. Bottom panel: RT-PCR analysis of actin mRNA.

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Supplementary Method: Production of anti-RORγ monoclonal antibody and immunohistochemical analysis. The hamster monoclonal antibody against murine RORγ was prepared at the Sloan Kettering Cancer Center monoclonal core facility. Animals were immunized with a His-tagged RORγ expressed in bacteria, and hybridoma supernatants were screened by ELISA on a MBP-RORγ fusion protein. Supernatants of positive clones were further screened for immunoblot reactivity with RORγin extracts from RORγ-transfected 293T cells and for immunofluorescence staining of thymic sections. Immunohistochemical localization of proteins was performed by incubating the slides in the presence of primary antibodies diluted in PBS, 0.1% Triton, 1% heat inactivated goat serum (HINGS) overnight at 4°C. Then sections were rinsed with PBS, 1% HINGS, and incubated with secondary antibodies 30 min at RT, rinsed in PBS, and cover slipped using Vectashield mounting medium (Vector Laboratories).